Excess dietary salt is a common cause of hypertension, but the mechanism(s) by which salt elevates blood pressure (BP) are unresolved. Extensive evidence indicates that salt retention promotes the secretion of endogenous ouabain (EO), an adrenocortical hormone. EO activates a Ca2+ signaling pathway in arterial smooth muscle cells (ASMCs) and neurons that augments vascular tone and elevates BP. This pathway involves a2/a3 Na+ pump inhibition, and increased Ca2+ entry via Na/Ca exchanger-1 (NCX1) and TRPC6 channels. Expression of these proteins is increased in several forms of hypertension, and is mimicked by chronic treatment of cultured ASMCs with ouabain but not digoxin. The aims of this project focus on acute and chronic ouabain-Na+ pump interactions and the specific role of Na+ pumps in the Ca2+ signaling pathway. Aim 1: To determine if in vivo arterial myocyte basal cytosolic [Ca2+] ([Ca2+]CYT), myogenic tone (MT), and agonist-evoked ASMC and endothelial responses are altered in mice with salt-sensitive hypertension and in mice with altered a2 Na+ pump expression. BP, and arterial [Ca2+]CYT and diameter will be measured simultaneously in vivo in anesthetized mice expressing a genetically-encoded Ca2+ biosensor in smooth muscle. The data will be correlated with parallel Ca2+ imaging and contraction experiments on isolated, pressurized small arteries from these mice. Aim 2: To determine the functional effects of Na+ pump ouabain high-affinity binding. The structure-activity relationship of several cardiotonic steroids (CTS) will be investigated by measuring their ability to mimic, or antagonize (as digoxin does), the action of nanomolar ouabain in raising [Ca2+]CYT, augmenting myogenic tone, and influencing BP in salt-dependent hypertension. Aim 3: To determine whether human (h)ASMC a2 Na+ pumps mediate ouabain-augmented Ca2+ signaling, as a2 does in rodents. We will test whether digoxin also augments Ca2+ signaling in freshly-dissociated hASMCs, and/or whether digoxin or other CTS antagonize the effect of ouabain on Ca2+ signaling. We also will determine the effects of chronic ouabain treatment on a2, NCX1 and TRPC6 protein expression in primary cultured hASMCs, and whether this effect is antagonized by digoxin and certain other CTS.